PT2086642E - Dnase for the treatment of male sub-fertility - Google Patents

Description

1

DESCRIPTION

" DNASE FOR MALE SUBFERTILITY TREATMENT " FIELD OF THE INVENTION

The invention relates to pharmacological compositions, and more specifically to those pharmacological compositions for use in the treatment of male subfertility. LIST OF THE PREVIOUS TECHNIQUE The following is a list of prior art references which is considered to be relevant to the understanding of the invention. The indication of these references in this document will sometimes be indicated by their list number in parentheses. 1. Isidori A, Latini M, Romanelli F. (1) Isidori A, Latini M, Romanelli F. Treatment of male infertility. Contraception Oct 2005; 72 (4): 314-8. 2. Kerin JFP, Peek J, Warms GM, Kirby C, Jeffrey R, Matthews CD, Cox LW (1984) Improved conception rate after intrauterine insemination of washed spermatozoa from poor quality semen. Lancet 1: 533-534. 3. Ombelet W, Puttemans P, Bosnians E (1995) Intrauterine insemination: a first step procedure in the algorithm of male subfertility treatment. Hum Reprod 10: 90-102. 4. Hinting A, Comhaire F, Vermeulen L, Dhont M, Vermeulen A, Vandekerckhove D (1990) Possibilities and limitations of techniques of assisted reproduction for the treatment of male infertility. Hum Reprod 5: 544-548. 5. Comhaire F, Milingos S, Liapi A, Gordts S, Campo R, Depypere H, Dhont M, Schoonjans F (1995) The effective cumulative pregnancy rate of different modes of treatment of male infertility. Andrology 27: 217-221. 2 6. Palermo GD, Cohen J, Alikani M, Adler A, Rozenwaks Z (1995) Intracytoplasmic sperm injection: a novel treatment for all forms of male infertility. Fertile Steril 63: 1231-1240. 7. Bartoov B, Berkovitz A, Eltes F. Selection of spermatozoa with normal nuclei to improve pregnancy rate with intracytoplasmic sperm injection. N Engl J Med. Oct. 4, 2001; 345 (14): 1067-8. 8. Bartoov B, Berkovitz A, Eltes F, Kogosovsky A, Yagoda A, Lederman H, Artzi S, Gross M, Barak Y. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertile Steril. Dec. 2003; 80 (6): 1413-9. 9. Berkovitz A, Eltes F, Lederman H, Peer S, Ellenbogen A, Feldberg B, Bartoov B. How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online. May 2006; 12 (5): 634-8. 10. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Fourth edition 1999. World Health Organization. Cambridge university press. 11. Aitken RJ. Sperm function tests and fertility. Int J Androl. Feb. 2006; 29 (1): 69-75; discussion 105-8. 12. Bartoov B, Berkovitz A, Eltes F, Kogosowski A, Menezo Y, Barak Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl. Jan.-Feb. 2002; 23 (1): 1-8. 13. Spadafora C. Sperm cells and foreign DNA: a controversial relation. Bioessays. Nov. 1998; 20 (11): 955-64. 14. Shak S, Capon DJ, Hellmiss R, Marsters SA, Baker CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci USA 1990; , 87: 9188-92.

BACKGROUND OF THE INVENTION

Currently, in the Western world, about one couple out of five is sub-fertile. Subfertility is defined as a failure to conceive after 1 year of unprotected intercourse. In about half of subfertile couples, male subfertility is observed, and in almost 40% of these couples, subfertility is only due to male factors. The male fertility status is evaluated by a semen analysis. The most common analysis, known as " routine semen analysis ", provides information on the number of spermatozoa present in the ejaculate, the proportion of sperm cells capable of moving (and / or progressively) and the percentage of cells which are morphologically normal. Male subfertility is usually associated with one or more of the low sperm production (oligozoospermia), weak sperm motility (asthenozoospermia), and abnormal sperm morphology (teratozoospermia). The combination of all three of these defects (" oligoastenoteratozoospermia " OTA) is the most common cause of male subfertility. The probability of conception increases with increased sperm concentration, motility and normal morphology. The World Health Organization (WHO) has proposed guidelines specifying the threshold values for these parameters to classify semen samples as normal or abnormal (10). The routine analysis of semen, however, has many limitations which result in a significant proportion of patients demonstrating unexplained subfertility. To refine the diagnosis, additional tests were developed which provide more information on the potential for fertilization of human semen. These tests include various functional tests of sperm cells (11), functional morphology tests such as the "organelle morphology examination of the sperm capable of moving" (MSOME, 12), and tests examining the extent of DNA damage in sperm cells, for example the sperm chromatin structure assay (SCSA, 11).

In some cases, the cause of male subfertility is known. These include varicoceles, hormonal disorders, infections, immunological infertility, obstruction of the male genital tract, and cryptorchism. Current medical and surgical therapies are available for these conditions (1). However, in a large number of cases of male subfertility, the cause is not known. In these cases, various treatments may be applied to the subfertile man, often on an ad hoc basis. These treatments include antiestrogens, aromatase inhibitors, androgens, FSH, pentoxifylline, arginine, camitine, glutathione, vitamins (A, C and E) and oligomineral (zinc, selenium), (I).

When a male infertility treatment does not lead to pregnancy after a reasonable period of time or if the diagnostic measures do not show that an improvement in fertility is possible, then assisted reproductive techniques (ART) may be considered. The various assisted reproduction procedures improve the fertilization potential of sperm by diverting some or all of the migration barriers of the upper and lower female genital tract as well as investment in the egg. ART procedures that are common now include intrauterine insemination (IU 1, 2, 3), classical in vitro fertilization (IVF, 4, 5) and IVF intracytoplasmic sperm injection (IVF-ICSI, 6). An improvement in ICSI is a technique known as " morphologically selected intracytoplasmic injection " (IMSI, 7-9) in which the fine morphology of spermatozoa capable of moving is examined by potent microscopy and the spermatozoa demonstrate the best morphology of the nuclei selected for ICSI.

Sperm cells of various species are known in vitro to bind to and absorb exogenous DNA (13). Binding to DNA is mediated at least in part by CD4 and MHCII molecules present on the surface of sperm cells and is antagonized by the IF-1 glycoprotein present in the seminal fluid. The interaction of sperm with extracellular acellular DNA activates intracellular nucleases such as DNase that cleave sperm genomic DNA, which eventually leads to a cell death process that resembles apoptosis. Bovine DNase I has been used clinically since 1965. It has been used before in the countries of the former Soviet Union to treat infections caused by DNA viruses such as Herpes and adenovirus. DNase is believed to degrade viral DNA in mono- and oligonucleotides. This medication is usually used to treat herpes simplex type 2 infection (genital herpes), Herpes simplex infections caused by herpes simplex, herpes zoster infections, inflammation of the respiratory tract such as bronchitis and pneumonia and tuberculosis. For these applications, it is given either by intramuscular injections, inhalation or eye drops. Recombinant human DNase I is another clinically used DNase I. It is an FDA approved medicine administered by inhalation to treat patients with cystic fibrosis (CF). In these patients, retention of viscous purulent secretions in the airways contributes to both reduced lung function and exacerbation of infections. Purulent pulmonary secretions contain very high concentrations of extracellular DNA released by degeneration of leukocytes that accumulate in response to infection. DNase I hydrolyzes DNA in the sputum of patients with CF and reduces viscoelasticity of sputum (14).

SUMMARY OF THE INVENTION The present invention is based on a new and unexpected discovery that high levels of acellular deoxyoligonucleotide present in the human blood circulation are associated with subfertility and that administration of exogenous acellular DNA reduces sperm quality and causes subfertility . The inventors thus found that providing subfertile male subjects with a DNase can improve semen quality and fertility potential.

According to the invention there is provided a pharmaceutical composition for treating male subfertility. The pharmaceutical composition of the invention comprises either an agent that blocks the effect of the acellular deoxyribonucleic acid molecules on the sperm cells. The agent is an enzyme that affects the level of acellular deoxyribonucleic acid molecules in body fluids. The enzyme is preferably a deoxyribonuclease (DNase). DNase can be an endodesoxyribonuclease or exodesoxyribonuclease (endodesoxyribonuclease cleaves within the molecules while exodesoxyribonuclease cleaves one end of the 3 'or 5' molecules).

A wide variety of DNases is known, which differs in their substrate specificities, chemical mechanisms, and biological functions. Non-limiting DNases that are applicable according to the invention are DNase I, DNase II, DNase-gamma, caspase-activated DNase (CAD), L-DNase II, DHPII. According to a preferred embodiment of the invention, DNase is DNase I. DNase may be of animal, plant, bacterial, viral, yeast or protozoan origin or may be recombinant DNase, preferably recombinant DNase, preferably recombinant human DNase . The composition may include, in combination with the agent, pharmaceutically acceptable carriers. By the term " pharmaceutically acceptable carrier " it is meant that any of the inert, non-toxic excipients which essentially do not react with the agent interfering with the DNA which has an effect on the sperm cells and is added thereto in order to give form or consistency to the composition and to provide protection against degradation of the agent and increase its survival outside and within the body and to achieve penetration within the body and distribution in the body fluids and to facilitate delivery of the agent in the subject's body and distribution to the target site (to DNA acellular).

According to one embodiment, the composition is in a form suitable for oral administration. According to another embodiment, the composition is in an appropriate form for injection, preferably intramuscular (IM), subcutaneous, or intravenous (i.v.) injections. According to yet another embodiment, the composition is in the form suitable for inhalation. Although other forms of administration are also applicable, it is noted that oral administration is a preferred route according to the invention because of improved adherence to the treatment.

Samples of semen obtained from men treated by the method of the invention can be used in conventional assisted reproduction (ART) technique. A non-limiting list of assisted reproduction techniques includes intrauterine insemination (IUI), in vitro fertilization (classical IVF); intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected injection (IMSI), as well as other techniques well known in the field of fertility. The agent used in the invention is administered and dosed according to good medical practice, taking into account the clinical condition of the male subject, the site and method of administration, the schedule of administration, the individual's age, body weight and other factors as well known to physicians. Doses may be single or multiple doses on a single day or in a period of several days / weeks / months (etc.). The treatment generally has an extent proportional to the severity of the condition of subfertility, efficacy of the agent and the male subject being treated. The dosage can be readily determined by administering to a plurality of tested individuals (individuals demonstrating subfertility) with various amounts of agent tested and then plotting the change in semen quality as a function of dosing. Alternatively, the dosage may be determined by experiments performed on appropriate animal models, and then extrapolating to humans using one of a plurality of conversion methods. As known, the amount that is considered to be effective in an individual may depend on a number of factors, such as the mode of administration (for example oral administration may require a higher dose to obtain a given plasma level of the agent than an intravenous administration); the age, weight, body surface area, health condition, and genetic factors of the individual, as well as the drugs to be administered simultaneously to the individual. The duration of the treatment will be determined according to the quantity and quality of the semen. Spermatogenesis occurs in the testicles and lasts approximately 64 days in humans. It is followed by 8-10 days of epididymal transport (EPT). The deleterious effect of acellular DNA is observed in different sections of the male genital tract: the seminiferous tubules of the testis and the tail epididymis. Thus, acellular DNA can affect different stages of spermatogenesis, producing damage to sperm cells manifested at different levels of severity. The damage to the testis is both quantitative and qualitative while epididymal damage is mainly qualitative. Thus, the duration of treatment may vary and should be determined according to the quantity and quality of the semen. The duration of the treatment is preferably selected within the following regimens:

Acute treatment A. 10 days. This course aims to reduce the effect of acellular DNA on sperm cells during epididymal transport.

B. 16 days. This course aims to reduce the effect of acellular DNA on sperm cells during the maturation phase of spermiogenesis and during epididymal transport.

Semi-chronic treatments C.

_26 days. This course aims to reduce the effect of acellular DNA on sperm cells during the spermatogenic cycle and during epididymal transport. D. 32 days. This course aims to reduce the effect of acellular DNA on sperm cells during complete spermiogenesis and during epididymal transport.

Chronic treatments E.

74 days. This course aims to reduce the effect of acellular DNA on sperm cells during complete spermatogenesis and during epididymal transport.

BRIEF DESCRIPTION OF THE DRAWINGS In order to understand the invention and to ascertain how it can be carried out in practice, a preferred embodiment will now be described, by way of non-limiting examples only, with reference to the accompanying drawings, in which: Figure 1 is a histogram showing DNA concentration in fertile and subfertile men; Figure 2 shows a histogram of the effect of DNA injections on the fertility potential of male mice; mice Figure 3 is a histogram showing the effect of acellular DNA injection on mice on the stability of chromatin in sperm cells; Figure 4 is a histogram showing the effect of acellular DNA injection on mice on the percentage of sperm cells expressing Fas receptor; Figure 5 is a histogram showing the effect of acellular DNA injection on mice on the activity of dandruff 3 in their sperm cells; Figure 6 is a histogram showing the effect of acellular DNA injection on mice on endogenous DNase activity in their sperm cells; Figure 7 shows morphological damage caused in testicular tissue by DNA injection; Figure 8 shows apoptotic damage caused in testicular tissue by DNA injections (TUNEL staining); Figure 9 shows the quantification of apoptotic damage caused in testicular tissue by DNA injections; Figure 10 shows morphological and apoptotic damage caused in testicular tissue by DNA injections (TUNEL staining); Figure 11 is a histogram showing the effect of intramuscular DNase injections on sperm density in semen of subfertile men; Figure 12 is a histogram showing the effect of intramuscular DNase injections on sperm motility in semen of subfertile men; Figure 13 is a histogram showing the effect of intramuscular DNase injections on progressive motility in the semen of subfertile men; Figure 14 is a histogram showing the effect of intramuscular DNase injections on the percentage of normal sperm cells in the semen of subfertile men as performed according to WHO using light microscopy; Figure 15 is a histogram showing the effect of intramuscular DNase injections on the percentage of sperm cells with different head defects in the semen of subfertile men as performed according to WHO using light microscopy; Figure 16 is a histogram showing the effect of intramuscular DNase injections on the percentage of sperm cells with amorphous heads in the semen of subfertile men as performed according to WHO using light microscopy; Figure 17 is a histogram showing the effect of intramuscular DNase injections on the percentage of sperm cells with defects of intermediate parts in the semen of subfertile men as performed according to WHO using light microscopy; Figure 18 is a histogram showing the effect of intramuscular DNase injections on chromatin stability in sperm cells in the semen of subfertile men; Figure 19 is a histogram showing the effect of intramuscular DNase injections on the percentage of sperm cells expressing the Fas receptor in the semen of subfertile men; Figure 20 is a histogram showing the effect of oral administration of DNase on sperm density; Figure 21 is a graph showing the effect of oral administration of DNase on semen volume; Figure 22 is a histogram showing the effect of oral administration of DNase on the concentration of sperm cells; Figure 23 is a graph showing the effect of oral administration of DNase on the percentage of sperm cells with normal nucleus morphology performed by MSOME; Figure 24 is a graph showing the effect of oral administration of DNase on the number of sperm cells with normal nucleus morphology performed by MSOME; Figure 25 is a histogram showing the effect of oral administration of DNase on the percentage of sperm cells with narrow-head morphology as performed by MSOME; Figure 26 is a histogram having the effect of oral administration of DNase on chromatin stability in sperm cells; Figure 27 is a graph showing the effect of oral administration of DNase on the percentage of sperm cells expressing the Fas receptor; Figure 28 is a graph showing the effect of DNase inhalation on the percentage of sperm cells with normal core morphology performed by MSOME; Figure 29 is a histogram showing the inhibition of DNase activity of internal sperm cells by the DNase citrate inhibitor; Figure 30 is a histogram showing the expression of the Fas receptor on the surface of sperm cells of fertile and subfertile men; Figure 31 is a histogram showing the activity of dandruff 3 in both sperm cells expressing the Fas receptor and sperm cells expressing the non-Fas receptor of subfertile men; Figure 32 is a histogram showing the number of offspring obtained in artificial insemination of mice using both sperm cells expressing the Fas receptor and sperm cells expressing the non-receptor

Fas; Figure 33 is a histogram showing the number of 2PN zygotes obtained in mouse insemination using both sperm cells expressing the Fas 13 receptor and sperm cells expressing the non-receptor

Fas; Figure 34 shows an embryonic development in mice after natural fertilization or after artificial insemination using both sperm cells expressing the Fas receptor and sperm cells expressing the non-Fas receptor; Figure 35 is a histogram showing the percentage of sperm cells expressing Fas receptor in the semen of subfertile men who both succeeded and failed in an IUI treatment; and Figure 36 is a histogram showing the activity of DNase in mouse blood plasma after administration of DNase.

EXAMPLES

Materials

Standard DNA solution; 100 mM calf thymus DNA were dissolved in double distilled water (DDW).

DPA solution: 1.5 g of diphenylamine were dissolved in 100 ml of glacial acetic acid and 1.5 ml of sulfuric acid were added. On the day of use, 0.5 ml of acetaldehyde was added.

Polyvinyl pyrrolidone (PVP) solution:

Medium PVP 1089000 obtained from Medi-Cult.

Kunitz DNase Activity Reaction Buffer:

5 mM MgSO 4 x 7H 2 O, 0.1 M Na Acetate pH 5.4 mg% calf thymus DNA. DNA and deoxyoligonucleotide levels (Burton method):

Standard DNA solution was diluted at 5, 10, 20 and 50 pg / ml in 166 μΐ. For each 166 μΐ plasma samples or standard DNA sample, 166 μΐ of HC104 IN and 664 μΐ of DPA solution were added. Samples were incubated at 37øC for 20 h, and then centrifuged for 10 min (15,000 g, 37øC), 300 μΐ of the supernatant were transferred to a 96-well plate and measured in a spectrophotometer at 600 nm.

Semen volume, sperm density and sperm concentration: The volume of each semen ejaculate was measured using a pipette. Sperm concentration was determined by counting the cells using both Helber's hemocytometer and Neubauer's hemocytometer (Helber is used when sperm concentration is high and Neubauer's when low). The sperm density (= number of cells in the total ejaculate) was calculated by multiplying the volume of ejaculate by the sperm concentration.

Sperm Motility and Progressive Motility: Sperm cells capable of or not moving were counted using both Helber's hemocytometer and Neubauer's hemocytometer and the percentage of sperm cells capable of moving accordingly was calculated. A sample of 20 random mobile sperm cells was examined for progressive motility by examining their ability to progress in a straight line up to 200 μπι and the percentage of sperm cell capable of progressively moving was consequently calculated.

Sperm morphology using the eosin-nigrosin staining technique: Sperm cells were stained using 2% eosin, a nuclear staining and 10% nigrosin for the background. 50 sperm cells were evaluated using light microscopy in an amplitude of 1000 x immersion oil. Analysis of sperm morphology was measured according to WHO guidelines (10).

Examination of the morphology of sperm organelles capable of moving in dry and moist (regular) (MSOME): 15

Preparation of sperm for morphological observation in wet (regular) MSOME:

A 1-2 μ al aliquot of the sperm suspension containing a few thousand sperm was transferred to a microbeater of sperm medium containing 0% -8% polyvinyl pyrrolidone (PVP) solution and placed in a glass bottom dish under oil of paraffin. The morphological evaluation of sperm cells in motion was made possible by the creation of small bundles extending from the rim of the droplets, which captured the heads of spermatozoa capable of moving.

Preparation of sperm for morphological observation in dry MSOME:

New post-liquefaction ejaculate was transferred to round bottom tubes at room temperature (22-25 ° C). The soft granules of ejaculated sperm cells were obtained by centrifugation in shaking vessels at 1500 RPM for 15 minutes at room temperature. Seminal plasma supernatant was removed. The sperm pellet was suspended in 1 ml IFV Ferticult medium (FertiPro N.V. Beemem, Belgium) and then re-centrifuged at 1500 RPM for 15 minutes at room temperature. The tubes were then transferred to a 5% CO2 incubator at 37øC during a swing incubation, tilted at 45ø. The supernatant was then carefully removed to the interface of the sperm pellet. The oscillating supernatant was centrifuged at 1500 RPM for 5 minutes at room temperature and the supernatant was discarded. The bead was wiped on microscope glass slides and air dried.

Sperm MSQME observation: Sperm cells were examined at 21øC by an inverted microscope (Olympus IX 70) equipped with Nomarski Optics, an objective Uplan Apo X 100 / 1.35 lens, and a 0.55 condenser lens AT. The images were captured by a DXC-950P (Sony) color video camera, which has a 3-inch HAD CCD power of 1.27 cm containing about 380,000 effective pixels for high-quality image production , and a color video monitor (Sony PVM-14M4E, HR-Trinitron). The morphological evaluation was conducted on the screen of the monitor, which, under the above configuration, reached a real amplitude of 6300. One hundred sperm motile of each sperm sample were examined for the morphological status of 6 subcellular organelles, post-acrosomal membrane, neck, mitochondria, tail and nucleus. The first 5 of these subcellular organelles were considered morphologically normal or abnormal based on the presence of specific malformations, which were defined as described in Bartoov et al., 1981;

Glezerman and Bartoov, 1993. For the nucleus, the morphological state was defined by both the form as chromatin content. The criteria for the core normally conformed by MSOME are smooth, symmetric, and oval configurations. The normal range of sperm core length and width was taken as 4.75 ± 0.28 μm and 3.28 ± 0.20 μm, respectively (standard deviation ± mean) while sperm cells outside this range were considered abnormal. The nuclear chromatin mass is homogeneous, with no regional nuclear disturbance and containing no more than one vehicle with borderline diameters of 0.78 + 0.18 μm from the frontal view. To be considered morphologically normal, a sperm nucleus must have both normal form and normal chromatin content. A sperm cell demonstrating a normal nucleus as well as a normal acrosome, post-acrosomal blade, neck, tail, mitochondria, and no droplet of cytoplasm or cytoplasm around the head was classified as morphologically normal.

Sperm Chromatin Structure Test (SCSA): SCSA determines the percentage of spermatozoa with 17

abnormal chromatin structure. Abnormal chromatin structure was defined as increased susceptibility of sperm DNA to acid-induced denaturation in situ. Amounts of DNA denaturation per cell were determined by flow cytometry that measured the shift of green fluorescence (native DNA) to red (denatured, single stranded DNA) in orange acridine colored nuclei. This shift is expressed as DFI, which is in the range of red to total fluorescence intensity (red + green), representing the amount of denatured single stranded DNA over the total cellular DNA. In the SCSA, DFI was calculated for each sperm in a sample, and the results were expressed as the average of 5,000 sperm cells. 100 μl aliquots of sperm cell suspension were mixed with 2 ml of buffer solution TNE (0.01 M Tris, 0.15 M NaCl, 0.001 M EDTA, pH 7.4) and centrifuged at 400 x gmax for 15 minutes at 25 ° C. The final pellet was resuspended in 2 ml of TNE buffer. The suspension was centrifuged at 3000 xmax for 15 minutes at 4 ° C. The obtained core beads were fixed by vigorous pipetting in a 500 μ fixação (acetone: 70% ethanol, 1: 1 v / v) fixative solution. All steps of the above procedure were performed at 4 ° C. The sample was centrifuged at 3000 x gmax for 15 minutes at room temperature and the bead was mixed with 100 μΐ acid detergent solution (0.08 M HCl, 0.15 M NaCl, 0.01% Triton X-100 , pH 1.2) for 30 sec at 4 ° C. The sample was then colored by adding 5 μΐ orange acridine (AO) staining solution containing 6 mg / ml AO. The sample was diluted with 500 μΐ citric buffer (citric acid, 0.037 M, 0.126 M Na2 HPO4, 0.001 M EDTA, 0.15 M NaCl, pH 6.0). Subsequently, the sample was subjected to flow cytometry on a Becton-Dickenson FACSort, San Francisco, CA, USA 18 flow cytometer equipped with a 15 mW argon ion laser ultrasonic laser with an excitation wavelength of 488 nm. The DFI ratio was calculated as a 1.1 ratio-time software package written by Jan van der Aa (Becton Dickenson, Erembodegem, Belgium) and with WinMDI 2.8 software. The DNA Fragmentation Index (DFI) is the mean fluorescence of sperm cells with nuclear DNA fragmentation. The% DNA Fragmentation Index (% DFI) is the percentage of sperm cells with nuclear DNA fragmentation. This parameter was computed based on the DFI distribution.

Activity of DNase in Intracellular and Blood Plasma: Sperm cells were subjected to three cycles of freezing and thawing and were then centrifuged for 10 min (27,000 g, 4øC). Plasma was separated from blood by centrifugation in EDTA tubes, 0.3 ml of supernatant was added to 0.6 ml of DNase activity buffer and the mixture was measured in a spectrophotometer at 260 nm against a reference which contained only buffer and medium . Calibration curves were made using a commercial bovine DNase I. The internal DNase of sperm cells was measured in Kunitz units. Since the Kunitz unit is defined as the amount of DNase which produces an increase of 1 O.D. per minute at 260 nm in a 1 cm pathway at pH 5 at 25 ° C in a reaction buffer containing 5 mM MgSO 4 x 7H 2 O, 0.1 M Na acetate pH 5 and 4 mg /% thymus DNA of calf. Plasma DNase was measured in Bartoov units. A Bartoov unit (BU) is defined as the amount of enzyme producing a ΔΑ26ο of 0.001 per minute in 1 cm path extension in 0H 7.5 at 25øC in a reaction buffer consisting of 0.1 M Tris , 10 mM CaCl2,

10 mM MnCl 2 and 0.05 mg / ml calf thymus DNA. Expression of the Fas receptor in sperm cells:

Sperm cells were washed with PBS and incubated in 4% paraformaldehyde (PFA) for 30 min at 25 ° C. The cells were then incubated for 15 min in blocking solution (1% BSA in PBS) and then incubated for 40 min with anti-goat Fas receptor antibodies. Anti-human Fas receptor antibody was FITC labeled and no second antibody was required. When mouse sperm cells were used, the cells were washed with PBS and incubated for 30 min with a second FITC-labeled anti-goat antibody. Cells were washed and fluorescence was measured on a Becton-Dickensen FASCSort flow cytometer.

Caspase-3 activity in sperm cells: The activity of caspase-3 was measured using a calorimetric kit obtained from R & D Company. Sperm cells were washed with PBS, incubated in lysis buffer for 10 min at 4 ° C and centrifuged for 1 min at 10,000 g. 50 μΐ reaction buffer and 5 μΐ caspase 3 colorimetric substrate were added to each 50 μΐ supernatant and incubated for 2 h at 37 ° C. The activity was measured on a spectrophotometer at 405 nm.

Separation of Sperm Cells Expressing Fas Receptor Expression of Non-Fas Receptor Expressing Sperm Cells: Sperm cells were washed with PBS and incubated for 30 min with anti-goat Fas receptor antibodies. The anti-human Fas receptor antibody was labeled with biotin and no second antibody was required. When mouse sperm cells were used, the cells were washed with PBS and incubated for a further 30 min with a second FITC-labeled anti-goat antibody. The cells were then washed with PBS and incubated for 20 min with either anti-biotin antibody conjugated to iron-containing microspheres (from Myltenyi biotech Company) for determination of the caspase activity, or anti-FITC antibody conjugated to these microspheres for mouse insemination. Separation of positive and negative sperm cells from the Fas receptor was performed as follows. Cells were washed with PBS and transferred through an immuno-magnetic MACS separation column from Myltenyi biotech Company. Unmarked sperm cells that did not express the Fas receptor flowed through the unbound column. Sperm cells expressing the Fas receptor were attached to the column and then eluted using plunger pressure.

Examination of DNase in plasma in mice after oral or by injection

C57 black mice were fasted for 12 hours and then received either oral PBS (placebo, n = 10) or oral administration of 9 mg DNase I in 200 μl PBS (n = 14), or intraperitoneal injection of 3 mg DNase I in 200 μl PBS (n = 6). Control mice were not treated at all (n = 10). Blood was collected from the heart of the mouse 40 min after injection or 1 h after oral administration. Plasma was separated from the blood by centrifugation in EDTA tubes and stored at -20 ° C. Plasma DNase activity was determined in Bartoov units.

Results

In vivo effects of acellular DNA

As noted in Fig. 1, endogenous acellular DNA levels are elevated in the blood of subfertile men compared to fertile men, indicating a correlation between high blood level DNA and subfertility. Of course, intravenous injections of acellular DNA into male mice impaired its fertility potential. Two groups of 10 male mice received intravenous injections of both 200æg of acellular DNA and PBS for 15 days. The mice were then mated to mice and the offspring number counted in both groups. Fig. 2 shows that mating with the control mice gave offspring of 6.9 ± 1.7 female while mating with mice injected with DNA gave proles of 0.7 ± 2.2 per female. In addition, intravenous injection of acellular DNA into mice impaired their nuclear sperm cell quality, as observed by a reduction in the stability of chromatin in sperm cells as expressed in an increase in DFI (Fig. 3). In addition, intravenous injection of acellular DNA into mice induced activation of apoptotic markers in the Fas receptor, caspase 3 and DNase within their sperm cells (Figs 4-6).

Acellular DNA injection affects not only the sperm cells but also the tissue of the testis. As seen in Figs. 7 and 10, acellular DNA caused the appearance of giant cells and depletion of the tubule epithelium by shifting the mantle from mature and non-mature testicular germ cells into the tubule lumen. Injection of acellular DNA also caused the appearance of large apoptotic areas (Figs 8-10). Spermatogenesis in the testicles takes approximately 64 days in humans. Thus, DNA damage to the testis is expected to have an impact on the development of sperm cells over a long period of time and repair of this data may require prolonged treatment.

Intramuscular administration of DNase I In order to examine whether DNase I can improve semen quality and fertility potential, it has been administered in subfertile men. In a group of 11 couples in which the male was previously identified as being subfertile and failed conceptually on IVF-ICSI treatment, the men received four times daily 25 mg of bovine DNase I by intramuscular (IM) injection for a period of 7-10 days. 7-11 days after the first injection, couples received a second ICSI treatment. Samples of semen and blood were taken from subjects twice before starting treatment and 4-7 and 8-15 days after the first injection and analyzed. As shown in Fig. 11, semen analysis revealed that sperm density increased by 70% after 8-22 15 days of treatment compared to cells (24.28 ± 14.73 x 106 / ejaculate pre-treatment versus cells 14.31 ± 10.15 x 106 / conjugate, respectively, P &lt; 0.08). In addition, a 60% increase in sperm motility was observed after 4-7 days of treatment compared to pretreatment (30.6 + 12.6% versus 19 + 11.3%, respectively, P &lt; 0, 033, Fig. 12) and a 76% increase was observed in progressive sperm motility after 8-15 days of treatment compared to pretreatment (47 ± 33.1% versus 26.7 ± 23.4%, respectively , P &lt; 0.010, Fig. 13). The morphology of sperm cells was analyzed according to WHO guidelines (10). After 8-15 days of DNase treatment, the percentage of morphologically shaped sperm cells in the semen of subjects was elevated by 129% compared to pretreatment (19.9 ± 12.1% vs. 8.7 ± 15.6 %, respectively, P &lt; 0.098, Fig. 14). In addition, DNase treatment was shown to cause a 24% reduction in the percentage of total head defects after 8-15 days of treatment compared to pretreatment (57.3 ± 17.6% versus 77.3+ 13 , 4%, respectively, P &lt; 0.015, Figure 15). In particular, a 73% reduction was observed in the percentage of amorphous heads after 8-15 days of treatment compared to pretreatment (9.4+ 7.3% versus 34.5+ 29.3% respectively, P &lt; 0.043, figure 16). In addition, a 60% reduction was observed in the percentage of defects of intermediate pieces after 8-15 days of treatment compared to pretreatment (5.4 + 5.1% versus 13.5 + 8.6%, respectively , P &lt; 0.040, Figure 17). The morphology of sperm cells was also analyzed by MSOME in 3 subjects. An improvement in a variety of MSOME parameters was observed for these individuals following DNase treatment (Table 1). In particular, there is an improvement in the percentage of normal nucleus sperm cells. ο -P 0 0) Ό Β α 0 < ^ rd Μ -μ * ιΗ LT) ω ο Τ3 0 0 μ &gt; μ <

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DNA damage in sperm cells correlates with impaired conception rates as well as to offspring health. One of the most widely used methods for evaluating this data is the &quot; chromatin structure test on sperm &quot; (SCSA), which measures the stability of chromatin in sperm in cells exposed to acid media. FIG. 18 shows that DNase treatment caused an increase in chromatin stability in sperm cells (lower% DFI values) after 4-7 days of treatment compared to pretreatment (15.1 ± 12.4% versus 25 ± 19 , 6%, respectively, P &lt; 0.033). In addition, DNase treatment caused a reduction in the Fas receptor expression of the apoptotic marker in sperm cell, both after 4-7 and 8-15 days of treatment, compared to pretreatment (26.5 ± 12.5% and 29.2 ± 17.4% versus 39.7 ± 23%, respectively, P &lt; 0.009 for 4-7 days and P &lt; 0.023 for 8-15 days, Figure 19).

Four women from 11 couples (36%) became pregnant after the second ICSI treatment following DNase treatment. The average pregnancy rate of a first ICSI cycle at the IVF center at which this attempt was conducted is 25% [range from 18% to 32%]. The pregnancy rate of a second cycle of ICSI from untreated patients at this IVF center is unknown since after an unsuccessful ICSI, they did not perform a second ICSI but instead were counseled about ICSI failures to donor insemination. In any case, the success rate of 36% of the second ICSI treatment is above 25% of the IVF center, so it is clear that DNase treatment has improved the chances of pregnancy of ICSI treatments.

The results in Figs. 11 to 19 and Table 1 show that intramuscular administration of DNase I improved semen in most semen quality parameters. Sperm density was improved by 70% at the end of the experiment. Sperm motility improved by 60% already after 4-7 days of treatment, but a reduction in motility occurred later, possibly due to an immune response that takes place around seventeen days and was observed after a fever of 24 h . Progressive sperm motility improved by 76% at the end of the experiment. Elevation in sperm motility and sperm density improved the fertility potential of the subjects and allowed those who could not succeed in the first ICSI treatment to conceive in a second ISCI treatment and possibly also in other ART treatments such as classic IVF or even an IUI treatment.

Administration of intramuscular DNase also improved sperm morphology as analyzed according to WHO guidelines (10). The percentage of normal sperm cells was increased by 129%. The percentage of sperm cells with defects in the head was reduced by 24% and led to normal (&lt; 65). The percentage of sperm cells with amorphous heads was reduced by 73% and the percentage of sperm cells with defects in the intermediate pieces was reduced by 60%. An improvement in sperm morphology was also observed using MSOME. The number of sperm cells considered for MSOME analysis and the number of normal nucleated sperm cells increased after treatment. The number of sperm cells with nuclear vacuoles, the number of narrowly sperm cells and the number of sperm cells with regional disturbance was reduced.

In addition, DNase treatment reduced sperm cell damage. Chromatin damage in sperm cells (as assessed by SCSA) was reduced by 40% already after 4-7 days of treatment and expression of the Fas receptor of the apoptotic marker was reduced by 26% after 4-7 days and by 33% after 8-15 days. The improvement in semen quality observed after treatment with DNase is expected to improve fertility potential and naturally an increase in the rate of success of ICSI treatment was observed after treatment.

Oral administration of DNase IA In order to examine whether oral administration of DNase I can improve semen quality as observed with intramuscular administration, a group of 9 men received 50 mg of DNase I orally four times daily for a period of 16 days. Samples of semen were taken and analyzed from subjects 5 days after starting treatment on the day of initiation of treatment and on days 5, 10 and 17 of treatment. As can be seen in Table 2, semen analysis revealed that sperm density (sperm / total ejaculate) significantly increased posttreatment compared to pretreatment (103 ± 50 x 106 cells / ejaculate versus 42 ± 38 x 106 cells / ejaculate, respectively, P &lt; 0.05). When the results were expressed as% control, an increase in sperm density was observed already 5 days of treatment and more dramatically after 10 and 17 days of treatment (235 ± 95%, 160 ± 72% and 284 ± 138% versus 100 % in control, respectively, P &lt; 0.05, Figure 20).

Table 2

Routine parameters -5 0 5 10 17 Volume (ml) 1.2 ± 0.5 1.4 ± 0.6 1.8 ± 0.7 1.7 ± 0.8 2.3 ± 0.6 ** Concentration (x 106 cells / ml) 31 ± 13 30 ± 12 42 ± 15 37 ± 12 45 ± 16 * Total spermatozoa (x 106 cells / ej) 37 ± 30 42 ± 38 75 ± 46 63 ± 52 103 ± 50 * Motility of sperm 85 ± 22 52 ± 5 61 ± 21 72 ± 12 86 ± 33% of sperm viability 76 ± 13 72 ± 11 71 ± 12 71 ± 12 11 ± 10 27

Routine parameters -5 0 5 10 17% normal forms 15 ± 8 11 ± 5 17 ± 16 18 ± 10 15 ± 9% of head defects 67 ± 10 70 ± 7 70 ± 16 12 ± 15 67 ± 9% defects of intermediate piece 6 ± 3 8 ± 4 6 ± 4 7 ± 2 6 ± 3% of tail defects 15 ± 10 12 ± 12 8 + 6 12 + 8 12 + 6 * p &lt; 0.05, n = 9 ** p &lt; 0.001, n = 9

In addition, an increase in semen volume was observed after 17 days of treatment (1.3 ± 0.5 ml pre-treatment versus 2.3 ± 0.6 ml after 17 days of treatment, P <0.001, Table 2 and Fig. 21). This increase indicates an increased function of the accessory glands. As shown in Table 2 and Fig. 22, the sperm concentration also increased during the treatment. MSOME analysis showed that the percentage of sperm cells having a normal nucleus increased 2-fold already after 5 and 10 days of treatment compared to pretreatment levels, as shown in Table 3 and Fig. 23. There was also a increase in the number of normal nucleated sperm cells after 10 and 17 days of treatment compared to pretreatment (5.0 ± 3.4 x 106 cells and 5.7 ± 5, 1 x 106 cells versus 1.6 + 2, 1 x 106 cells , respectively, P &lt; 0.05, Table 3 and Fig. 24). In addition, a decrease in sperm cells with narrow heads was observed after 5, 10 and 17 days of treatment as a control (84 ± 15%, 85 ± 15% and 83 ± 21% versus 100% in the control, respectively, P &lt; 0.05, Fig. 25).

Table 3 28

MSOME parameters -5 0 5 10 17% core normal 5.2 + 2.7.7 ± 2.06 9.0 ± 4.3 * 10 ± 6.1 ± 12.3 ± 6.3 ** Normal nucleus (x106 cells) 2.0 ± 2, 7 1.2 ± 0.9 ± 3.0 ± 2.4 ± 4.5 ± 3.4 ± 5.7 ± 5 ± 1 * Total abnormal forms 159 ± 31 154 ± 30 154 ± 30 150 ± 28 151 ± 30% Nuclear vacuoles 80 ± 13 79 ± 14 79 ± 12 73 t 13 11 ± 10% of small forms 0.1 ± 0.3 ± 0.5 0.4 ± 1.2 0.1 ± 0.3 0.4 ± 0.6% of large forms 3.3 ± 2.3 ± 4.4 ± 2.3 ± 2.9 ± 2.2 ± 1.4 2.5 ± 2.3% narrow forms 40 ± 10 41 ± 14 33 ± 10 35 t 9 34 ± 9% broad forms 4.2 ± 3.0 ± 3.2 ± 2.4 3.5 ± 2, 0 5.2 ± 4.0 2.9 ± 2.0% regional disturbances 31 ± 7 25 ± 11 34 ± 8 34 t 9 34 ± 8 vacuoles index 1.8 ± 0.3 0.7 ± 0.02 1.6 ± 0.3 1.7 ± 0.3 * p <0.05, n = 9 ** P <0.001, n = 9

An increase in chromatin stability in sperm cells (smaller DFI values) was observed already after 5 days of treatment as a control (75 ± 36% versus 100%, respectively, P &lt; 0.05, Fig. 26). This increase was more pronounced after 10 and 17 days (65 ± 31% and 62 ± 33% versus 100%, respectively, P &lt; 0.005, Fig. 26). In addition, the percentage of sperm cells expressing the apoptotic marker Fas receptor decreased after 10 days of treatment compared to pretreatment (10.6 ± 7.5% versus 35.4 ± 26.4%, respectively, P &lt; 0.05, Fig. 27). A further increase was observed after 17 days of treatment compared to pretreatment (6.4 ± 5.2% versus 35.4 ± 26.4%, respectively, P &lt; 0.005, Fig. 27).

Among those individuals who participated in the orally administered DNase trial, only one individual attempted with their female partner to successfully complete the pregnancy. This couple had previously failed in four procedures with IUI. After DNase treatment, the couple successfully achieved spontaneous pregnancy. 29

The results in Figs. 20 to 27 and Tables 2 and 3 show that oral administration of DNase I has the following effects: 1. Increase the volume of semen. 2. Increase the density and concentration of sperm cells. 3. Improve sperm quality by increasing the fraction of sperm cells that has a normal nucleus, increasing chromatin stability and decreasing the ratio of cells expressing the Fas receptor. In order to examine whether administration of DNase by inhalation leads to similar effects on semen quality, it was examined for one case. In the inhalation procedure, the subject inhaled 3 times a day 8 mg of the enzyme, taken at 4-hour intervals. Inhalation of the enzyme leads to an increase in the percentage of abnormal nuclei that was deeper after 9 and 17 days of inhalation (Fig. 28).

Inhibition of DNase in Endogenous Sperm Cells In order to examine whether DNase inhibitor citrate can inhibit DNase in intracellular sperm cells, sperm cells were incubated with or without 10 mM citrate for up to 8 hours. Intracellular DNase activity was measured hourly and the sum of DNase activity was calculated. The sum of the DNase activity in the citrate treated sperm cells was 43% of the control sperm cells (Fig. 29), indicating inhibition of intracellular DNase activity by citrate. The relationship between Fas receptor expression and caspase 3 activity in sperm and fertility cells

One of the known apoptosis markers is the expression of the Fas receptor. As seen in Fig. 4, injection of acellular DNA into mice leads to a statistically significant increase in the expression of the Fas receptor on the membrane 30 of its sperm cells. In vitro incubation of sperm cells with acellular DNA also leads to significant elevation in Fas receptor expression on the sperm cell membrane (not shown). In order to determine if there is a correlation between Fas receptor expression and male fertility, Fas receptor expression on the surface of sperm cells was determined in fertile and subfertile men. The percentage of sperm cells expressing the Fas receptor was four times higher in subfertile than in fertile (78.8 ± 27.4 versus 12.7 ± 6.4, P &lt; 0.001, Fig. 30). Next, in order to examine whether there is a correlation between Fas receptor expression in subfertile men sperm cells and caspase-3 activity in the apoptotic marker, sperm cells from subfertile men were separated according to their expression of receptor Fas. Caspase-3 activity was determined in each sperm cell population. The percentage of caspase-3 activity in Fas receptor-positive sperm cells was 4-fold higher than caspase-3 activity in the Fas receptor negative subpopulation (207.2 ± 39.6 versus 47.2 ± 10.5 , P &lt; 0.001,

Fig. 31). In order to determine if Fas receptor expression correlates with fertility potential, the following experiment was conducted: mouse sperm cells were incubated with acellular DNA (which was found to stimulate expression of the Fas receptor) and then cells from sperm expressing the Fas receptor were separated from cells that did not express the Fas receptor. Four groups of 10 mice were inseminated either with sperm cells expressing Fas receptor (Fas +), sperm cells expressing Fas receptor (Fas-), sperm cells that were incubated with acellular DNA but not classified according to expression of the Fas receptor, and control cells that were not exposed to DNA. All 31 inseminations were performed with the same number of sperm cells. As can be seen in Fig. 32, no fetus was obtained from insemination with sperm cells (Fas-) in contrast to sperm cells (Fas +) or control sperm cells which produced 3 ± 1.4 and 2.7 ± 1.1 average fetuses per mouse, respectively. Insemination with cells that were incubated with acellular but unclassified DNA gave rise to only 0.3 ± 0.4 average fetuses per female. In order to determine the stage at which sperm cells expressing receptor Fas failed to produce offspring, four groups of mice were inseminated with sperm cells as described above. 16-20 hours after insemination, females were sacrificed, oocytes were taken from the oviduct and the number of 2 zygotes pronucleus (2PN) was counted. As shown in Fig. 33, Fas (+) and Fas (-) sperm cells gave rise to a similar number of 2PN zygotes and no significant difference was observed between control sperm cells and sperm cells that were incubated with Acellular DNA. Then, a similar insemination was performed using Fas (+) and Fas (-) sperm cells and the mice were sacrificed at different stages of pregnancy. Embryonic development was examined in both treatments and compared as embryonic development in natural fertilization. No embryonic development beyond the 2PN stage was observed in females inseminated with Fas (+) sperm cells while females inseminated with Fas (-) sperm cells demonstrated normal embryonic development as in natural fertilization (Fig. 34).

In addition, the correlation between Fas receptor expression in sperm cells and the outcome of IUI treatment was examined in humans. Semen samples from 72 subfertile couples who underwent IUI treatment were examined for Fas expression by fluorescence staining with anti-human Fas receptor and FACS analysis. 18 cases resulted in pregnancy while the remaining 54 cases did not result in pregnancy. As shown in Fig. 35, the frequency of Fas (+) sperm cells in the group that obtained pregnancy (22.4 ± 18.2 versus 33.1% ± 16.6, respectively, P &lt; 0.027.

DNase activity in mouse blood plasma In order to examine the ability of DNase to reach the bloodstream upon oral administration, the mice were treated with DNase either by intraperitoneal injection or oral administration and DNase activity was examined after 1 hour. As shown in Fig. 36, a significant increase in DNase activity in blood plasma was observed in both the peritoneally injected and orally administered groups compared to the negative control group (14399 ± 4000 and 10853 ± 2150, respectively, versus 1345 ± 580 , p <0.001). A small increase in DNase activity was also observed in the placebo group (4055 ± 3500).

An animal study examining the safety and efficacy of DNase

An animal study examining the safety and efficacy of DNase in rodents and dogs was conducted at the Federal Institute of Toxicology in Russia. The study showed that the acute and prolonged administration of DNase does not have a toxic effect on the studied warm-blooded laboratory animals. Administration of DNase daily, prolonged (30 days) in experimental animals at doses that were over 100 times higher than the recommended human dose did not produce detectable deleterious effects on major body systems (nervous, cardiovascular, hematopoietic, secretory , respiratory), metabolism, general health condition, development and basic homeostatic parameters of the organism. The absence of irritant effects on the digestive system when DNase administration was also observed. 33

DOCUMENTS REFERRED TO IN THE DESCRIPTION

This list of documents referred to by the author of the present patent application has been prepared solely for the reader's information. It is not an integral part of the European patent document. Notwithstanding careful preparation, the IEP assumes no responsibility for any errors or omissions.

Non-patent literature referred to in the description • ISIDORI A; LATINI M; ROMANELLI F. Isidori A, Latini M, Romanelli F. Treatment of male infertility. Contraception, October 2005, vol. 72 (4), 314-8 [0002] • KERIN JFP; PEEK J; WARMS GM; KIRBY C; JEFFREY R; MATTHEWS CD; COX LW. Improved conception rate after intrauterine insemination of washed spermatozoa from men with poor quality semen. Lancet, 1984, vol. 1, 533-534 [0002] • OMBELET W; PUTTEMANS P; BOSMANS E. Intrauterine insemination: a first step procedure in the algorithm of male subfertility treatment. Hum Reprod, 1995, vol. 10, 90-102 [0002] • HINTING A; COMHAIRE F; VERMEULEN L; DHONT M; VERMEULEN A; VANDEKERCKHOVE D. Possibilities and limitations of assisted reproduction techniques for the treatment of male infertility. Hum Reprod, 1990, vol. 5, 544-548 [0002]

• COMHAIRE F; MILINGOS S; LIAPI A; GORDTS S; FIELD R; DEPYPERE H; DHONT M; SCHOONJANS F. The effective cumulative pregnancy rate of different modes of treatment of male infertility. Andrology, 1995, vol. 27, 217-221 [0002]

• PALERMO GD; COHEN J; ALIKANI M; ADLER A; ROZENWAKS Z. Intracytoplasmic sperm injection: a novel treatment for all forms of male infertility. Fertile 34

Steril, 1995, vol. 63, 1231-1240 BARTOOV B; BERKOVITZ A; ELTES F. Selection of spermatozoa with normal nuclei to improve pregnancy rate with intracytoplasmic sperm injection. N Engl J Med., 04 Oct. 2001, vol. 345 (14), 1067-8 BARTOOV B; BERKOVITZ A; ELTES F; KOGOSOVSKY A; YAGODA A; LEDERMAN H; ARTZI S; GROSS M; BARAK Y. Pregnancy rates are higher with intracytoplasmic morphologically selected sperm injection than with conventional intracytoplasmic injection. Fertile Steril., December 2003, vol. 80 (6), 1413-9 [0002] • BERKOVITZ A; ELTES F; LEDERMAN H; PEER S; ELLENBOGEN A; FELDBERG B; BARTOOV B. How to improve IVF-ICSI outcome by sperm selection. Reprod Biomed Online, May 2006, vol. 12 (5), 634-8 [0002] • WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. Cambridge University Press, 1999 [0002] • AITKEN RJ. Sperm function tests and fertility. Int J Androl., February 2006, vol. 29 (1), 69-75 [0002] • BARTOOV B; BERKOVITZ A; ELTES F; KOGOSOWSKI A; MENEZO Y; BARAK Y. Real-time fine morphology of motile human sperm cells is associated with IVF-ICSI outcome. J Androl., January 2002, vol. 23 (1), 1-8 [0002] • SPADAFORA C. Sperm cells and foreign DNA: a controversial relation. Bioessays, November 1998, vol. 20 (11), 955-64 [0002]

• SHAK S; CAPON DJ; HELLMISS R; MARSTERS SA; BAKER CL. Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum. Proc Natl Acad Sci USA, 1990, vol. 87, 9188-92 [0002]

Claims (5)

A pharmaceutical composition for use in the treatment of male subfertility comprising a deoxyribonuclease (DNase) and a physiologically acceptable carrier. The composition for use according to claim 1, wherein the enzyme is of animal, vegetable, bacterial, viral, yeast, or protozoan origin or is a recombinant enzyme or a recombinant human enzyme. The composition for use according to claim 1 or 2, in a form suitable for oral administration. The composition for use according to claim 1 or 2, in a pharmaceutical form suitable for administration by inhalation. The composition for use according to claim 1 or 2, in a pharmaceutical form suitable for administration by injection.